Ion channels are instrumental in the generation of membrane potential, receptor potential, and action potential. They are the molecular building blocks for what is an essential characteristic of many cells in neuroscience: excitability. These proteins are implicated in the physiology and pathophysiology of all excitable tissues, underlie many disease processes including epilepsies and arrhythmias, and are major targets of essential drugs used in clinical medicine. Potassium channels are the phylogenetic founders of a large superfamily of structurally related ion channels that includes nucleotide gated channels, sodium channels, and calcium channels. Potassium channels are typically assembled from four identical subunits in a four-fold symmetrical fashion. This rather simple structural blueprint and the substantial practical advantage of only one subunit type have made potassium channels a much studied model system. This proposal explores two aspects of potassium channels: drug binding and voltage- dependence. In aim 1 we will determine the structural basis of the drug-channel interaction. In aim 2 we will attempt to describe the voltage-sensing domain of the channel in its native form. We will use a combination of cysteine mutagenesis, biochemistry, electrophysiology, site-directed spin labeling, and X-ray crystallography to study two prokaryotic potassium channels: KcsA and KvAP. The long-term goal of this proposal is to understand functional properties of ion channels at a structural level. PUBLIC HEALTH RELEVANCE: Ion channels are membrane proteins that allow ions to pass from one side of the cell membrane to the other. They are the molecular hardware that generates all electrical signals in the human body. These signals are used to coordinate the beating of the heart and underlie the complex function of the central and peripheral nervous system. When ion channels malfunction, serious maladies ensue, such as heart arrhythmias, epilepsy, and even death. Ion channels are also important drug targets. Drugs effecting ion channels are used every day in clinical medicine and are often implicated in serious drug side effects. Determining the precise structure and mechanism of action of these channels will allow for the development of safer and more effective drugs.